Biological waste material from industry including agriculture contains sugars and their derivatives such as sugar acids. The conversion of such waste to useful products has been of interest and challenge in the field of biotechnology for a long time. D-galacturonic acid is the major component of pectin, a low price raw material enriched e.g. in sugar beet pulp, and a carbon source for microorganisms living on decaying plant material.
For bacteria a pathway is known consisting of 5 enzymes converting D-galacturonic acid (D-galacturonate) to pyruvate and D-glyceraldehyde 3-phosphate (FIG. 1). The intermediate metabolites are D-tagaturonate, D-altronate, D-erythro-3-deoxy-hexulosonic acid (2-keto-3-deoxy D-gluconate) and D-erythro-3-deoxy-hexulosonic acid 6-phosphate (2-keto-3-deoxy D-gluconate 6-phosphate). The enzymes are uronate isomerase (EC 5.3.1.12), an NADH utilizing D-tagaturonate reductase (EC 1.1.1.5), altronate dehydratase (EC 4.2.1.7), 2-keto-3-deoxy D-gluconatekinase (EC 2.7.1.45) and 2-keto-3-deoxy D-gluconate 6-phosphatealdolase (EC 4.1.2.14).
The pathway of FIG. 1 has only been described tier prokaryotic organisms, i.e. there are no reports about a similar pathway in eukaryotic microorganisms. A pathway must exist in eukaryotic microorganisms, since many species of yeast and mould can utilize and grow on D-galacturonate, however very little is known about such a pathway.
There are only a few studies on D-galacturonic acid catabolism in eukaryotic microorganisms. Uitzetter et al. 1986 (J. Gen. Microbial., 132, 1167-1172) mutagenized the filamentous fungus Aspergillus nidulans and found that mutants lacking pyruvate dehydrogenase or pyruvate carboxylase activity were unable to grow on D-galacturonate, whereas a pyruvate kinase mutant was able to grow on D-galacturonate. This was interpreted to indicate that D-galacturonate is converted to pyruvate but not through phosphoenolpyruvate, i.e. this would be similar to the case in bacteria. Visser et al. (1988) J. Gen. Microbial., 134:655-659), speculated that in A. nidulans D-galacturonic acid is catabolized through glyceraldehyde and pyruvate, which differs from the bacterial pathway in that the bacteria metabolize it through D-glyceraldehyde 3-phospate. It has further been suggested that D-galacturonic acid is metabolized through glycerol, since a glycerol kinase mutant had reduced growth on D-galacturonic acid (Witteveen, C. F. et al., (1990) J. Gen. Microbial., 136:1299-1305), and an NADP dependent glycerol dehydrogenase was induced by D-galactronic acid (Sealy-Lewis, H. M. and Fairhurst, V., (1992) Curr. Genet., 22:293-296).
There are no reports about genes, which are similar to the genes of the bacterial D-galacturonic acid pathway as shown in FIG. 1 in the genome of any eukaryotic microorganism of which the genome was sequenced. This suggests that there is a eukaryotic path for the catabolism of D-galacturonic acid, which is different from the bacterial path.
In fungi D-galacturonic acid has been suggested to be converted into galactonate by an aldoketo reductase, after which a dehydratase or racemase modifies galactonate to 2-keto-3-deoxygalactonate, and an aldolase splits 2-keto-3-deoxygalactonate into pyruvate and glyceraldehyde. Martens-Uzunova, E. et al., (Fungal Genetics Newsletter, vol. 52, Supplement (185), XXIII Fungal Genetics Conference Mar. 15-20, 2005, Pacific Grove, Calif.) have identified a cluster of co-expressed genes that encode the necessary putative aldoketo reductase, racemase and aldolase. No dehydratase was identified, nor do the authors explain the role of the racemase. In fact they do not mention whether said galactonate or said 2-keto-3-deoxygalactonate or said glyceraldehyde is in L- or D-configuration.
The present invention is based on finding a novel gene and enzyme involved in the fungal metabolism of D-galacturonic acid. This finding reveals a putative metabolic pathway of D-galacturonic acid. DNA comprising the gene may be used to produce genetically modified microorganisms, which are capable of effectively fermenting carbohydrates and their derivatives, such as sugar acids and their derivatives, from a biomaterial to obtain useful fermentation products, such as ethanol.
One aim of the invention is to provide an enzyme protein, which can be expressed by a host for the conversion of sugar acids and their derivatives to useful conversion products in a fermentation medium, or which is in the form of an enzymatic preparation for in vitro conversion of sugar acids and their derivatives to useful end products or intermediate products.
Another aim of the invention is to provide a genetically modified organism in which the expression of the gene is prevented, and which therefore is capable of accumulating the substrate of this enzyme.
The novel DNA molecule encodes a sugar acid dehydratase that is active on sugar acids, where the hydroxyl group of C2 is in L and the hydroxyl group of C3 is in D configuration in the Fischer projection. The enzyme does not exhibit activity with sugar acids, where the hydroxyl group of C2 is in D and the hydroxyl group of C3 is in L configuration. Such dehydratases are previously known e.g. from Niu et al. (J. Am. Chem. Soc., (2003) 125:12998-12999), who described a dehydratase which is active on L-arabonic acid and D-xylonic acid. Another example is the D-gluconate dehydratase that is active in the non-phosphorylated Entner-Doudoroff pathway (see e.g. Buchanan et al. (1999) Biochem. J., 343:563-570).
In a crude extract of the bacterium Pseudomonas saccharophila enzyme activity converting D-arabonic acid has been found, and the reaction product was believed to be 2-keto-3-deoxy-D-arabonic acid (Palleroni, N. J. and Doudoroff, M., (1956) J. Biol. Chem., 223:499-508). However, no gene was isolated nor expressed.